IL-2 is a soluble protein which is capable of modulating lymphocyte reactivity and promoting the long-term in vitro culture of antigen-specific effector T-lymphocytes (mitogenesis) and, in the past, has been produced by stimulating mouse, rat or human lymphocyte cells with a mitogen. For instance, Morgan et al. in "Selective in vitro Growth of T Lymphocytes from Normal Human Bone Marrows", 193 Science 1007 (1976) and Ruscetti et al. in "Functional and Morphological Characterization of Human T Cells Continuously Grown in vitro", 119 The Journal of Immunology 131 (1977), both discussed a process for culturing pooled normal human lymphocytes in Roswell Park Memorial Institute (hereafter "RPMI") medium containing autologous serum and the mitogen phytohemagglutinin (hereafter "PHA").
Gillis and Smith, in "Long Term Culture of Tumor-Specific Cytotoxic T Cells", 268 Nature 154 (1977) reported preparing murine IL-2 by stimulating normal DBA/2 mouse spleen cells with the mitogen concanavalin A (hereafter "Con A") in an RPMI 1640 culture medium containing fetal calf serum (hereafter "FCS").
Farrar et al. in "Biological Relationship of Thymocyte Mitogenic Factor and Factors Enhancing Humoral and Cell-Mediated Immune Responses", 121 The Journal of Immunology 1353 (1978), also disclosed preparing IL-2 from murine spleen cells incubated with Con A in a tissue culture medium containing normal mouse serum (hereafter "NMS").
Gillis et al. reported generating IL-2 from murine and rat spleen cells cultured in a RPMI 1640 tissue culture medium supplemented with heat-inactivated FCS, penicillin-G, and gentamycin. The murine and rat spleen cells were stimulated by various mitogens including Con A, PHA, and pokeweed mitogen (hereafter "PKM"), "T-Cell Growth Factor: Parameters of Production and a Quantative Microassay for Activity", 120 The Journal of Immunology 2027 (1978).
IL-2 has also been prepared from human peripheral blood mononuclear cells by culturing the cells in RPMI 1640 medium supplemented with autologous human serum, penicillin, gentamycin, fresh L-glutamine, and PHA. Gillis et al., "Biochemical Characterization of Lymphocyte Regulatory Molecules-II. Purification of a Class of Rat and Human Lymphokines", 124 The Journal of Immunology 1954 (1980).
Gillis et al. in "Biochemical and Biological Characterization of Lymphocyte Regulatory Molecules-III. The Isolation and Phenotypic Characterization of Interleukin-2 Producing T Cell Lymphomas", 125 The Journal of Immunology, 2570, (1980), identified the preparation of IL-2 from T cell leukemia and lymphoma cell lines, specifically a radiation-induced splenic lymphoma from the B10.BR mouse (LBRM-33) cultured in RPMI 1640 supplemented with heat inactivated FCS, 2.5.times.10.sup.-5 M 2-mercaptoethanol, N-2-hydroxy-piperazine-XI.sup.1 -2-ethene-sulfonic acid (hereafter "Hepes") buffer, penicillin, streptomycin and fresh L-glutamine. The cultures were stimulated with various mitogens including Con. A, and PHA.
IL-2 purified from these mouse, rat and human normal T-lymphocytes, has been found to retain different types of biological activity, including: (1) marked enhancement of thymocyte mitogenesis, Watson et al., "Biochemical and Biological Characterization of Lymphocyte Regulatory Molecules-I. Purification of a Class of Murine Lymphokines", 150 Journal of Experimental Medicine 849, (1979) and Gillis et al. supra, 124 Journal of Immunology 1954 (1980); (2) promotion of long term in vitro proliferation of antigen specific helper or killer T cell lines, Gillis et al., supra, 268 Nature 154 (1977) and Watson, "Continuous Proliferation of Murine Antigen Specific Helper T Lymphocytes in Culture", 150 Journal of Experimental Medicine 1510 (1979); and (3) induction of cytotoxic T lymphocyte (hereafter "CTL") reactivity and plaque-forming cell responses in cultures of nude mouse spleen cells. Watson et al., supra, 150 Journal of Experimental Medicine 849 (1979) and Gillis et al., supra, 124 The Journal of Immunology 1954 (1980). Accordingly, these identified biological activities of IL-2 indicate that IL-2 is useful in elevating immune responses and restoring immune deficient T cell populations (nude mouse spleen cells) to normal levels of cell and humoral immunity. Furthermore, these results suggest that IL-2 production and response are important parameters of immunological functions which may be useful in clinical diagnosis of aberrant immunity. Moreover, the fact that human IL-2 makes possible the in vitro proliferation of antigen specific human, mouse and rat killer T cells emphasizes the importance of human IL-2 as a research reagent.
The above cited articles by Morgan et al., 193 Science 1007 (1976); Ruscetti et al., 119 The Journal of Immunology 131 (1977); and Gillis et al., 124 The Journal of Immunology 1954 (1980), discuss production of human IL-2 from lectin stimulated human splenic and peripheral blood lymphocyte conditioned media. However, these production sources and techniques result in weak concentrations of IL-2, with purification of IL-2 requiring fractionation of large volumes of conditioned media containing IL-2 in order to obtain only very small quantities of human IL-2 activity. As a consequence, sufficient quantities of concentrated human IL-2 have not been available for in vivo experiments, nor to study effectively the final molecular characterization of this lymphocyte regulatory molecule.
U.S. patent application Ser. No. 249,905, filed Apr. 20, 1981 entitled "Process For Preparing Human Interleukin 2" inventor Steven Gillis describes the production of human IL-2 by induction of a malignant neoplastic cell line such as the Jurkat-FHCRC line with a T-cell mitogen such as phytohemagglutinin (PHA) optionally in the presence of a phorbol ester such as phorbol myristate (PMA). Partial purification of the IL-2 was achieved by a procedure involving ammonium sulfate precipitation/dialysis, gel filtration chromatography (Sephadex G-100), ion exchange chromatography DEAE cellulose), flat bed isoelectric focusing and analytical SDS polyacrylamide gel electrophoresis. The majority of IL-2 biological activity was electrophoretically eluted from the protein band (one of nine to sixteen separate bands observed on the gel) having a molecular weight of approximately 14,000 daltons. See also Frank et al. J. Immunol. 127, 2361 (1981) and Watson et al. Lymphokines 6, 95 (1982) for a corresponding disclosure.
Mier and Gallo, J. Immunol. 128, 1122 (1982) and also Lymphokines 6, 137 (1982) have reported on the purification of IL-2 from normal lymphocytes to provide "a nearly homogeneous material" using preparative SDS gel electrophoresis as the last step after a sequence of anion exchange chromatography and gel filtration procedures. Their product had a molecular weight of 13,000 on SDS-PAGE and 20-25,000 on gel filtration and an isolectric point of 6.8. This material was very unstable even at -70.degree. C. and required addition of BSA or polyethylene glycol to retain activity.
Stadler and Oppenheim, Lymphokines 6, 117 (1982) describe IL-2 purified from peripheral blood mononuclear cells, tonsil and spleen cells which exhibited charge heterogeneity after purification on column chromatography, gel filtration and electrofocusing. Three charge species having PI's of 6.5, 7.2 and 8.2 were found and the heterogenecity possibly attributed to differences in the degree of glycosylation. Reference is made on page 127 to the observation of Robb and Smith of a single charge species of IL-2 derived from the Jurkat cell line having a pI of 8.2 (Mol. Immunol. 1981 indicated to be in press).